Severe acute respiratory syndrome is a life-threatening human illness characterized by mortality rates exceeding 50% in the elderly. The SARS coronavirus contains a approximately 30Kb single-stranded, positive polarity RNA genome. The availability of a full-length infectious cDNA of the SARS genome would not only provide complete genetic control over the virus, but allow for rational design of live viruses as candidate vaccines. Consequently, we believe that a SARS reverse genetic system must rank near the top of the priorities for controlling this important human pathogen. We are the only group in the US to have successfully assembled full-length infectious cDNAs of the coronaviruses, mouse hepatitis virus (MHV) and transmissible gastroenteritis virus (TGEV) and have demonstrated that these full length constructs provide novel opportunities for studying the genetics of coronavirus replication and pathogenesis. In aim 1, we will develop a full length SARS cDNA clone and compare the phenotype of rescued molecular cloned viruses with wildtype using biochemical assays and macaque challenge experiments. In Aim 2, we will develop high titer SARS single hit replicons for use as expression vectors and vaccines. In aim 3, we will select for SARS host range mutants that replicate in murine cells, identify the mechanism of SARS cross species transmission using reverse genetic approaches and evaluate the pathogenicity of these viruses in rodents and non human primates. The goal of this application is to establish genetic control over the SARS genome and provide uniform reagents that will be used by other groups throughout the country.